Progress during the second grant period characterized the involvement of a number of signaling pathways that control induction, left-right asymmetry and early patterning of the heart. This renewal application will focus on the heart-inducing pathways with the objective of defining diffusible factors that specify cardiomyogenesis and promote differentiation of cardiomyocytes. Our point of departure is knowledge of several signaling pathways that stimulate cardiogenesis in embryos and stem cells. The first pathway is initiated by Dickkopf-1 (Dkk1) or other canonical Wnt/beta-catenin signaling antagonists. This pathway involves the divergent homeodomain protein Hex as an essential regulator of as yet unidentified diffusible factors. We present evidence that Dkk1 intiates a second, unexpected pathway that is essential for progression to later stages of myocardial differentiation and morphogenesis. The third pathway is initiated by the TGF-beta family member Nodal while a fourth pathway is non-canonical Wnt signaling. An unexpected finding of our research is that both Dkk1 and Nodal induce heart tissue indirectly, by acting on foregut endoderm to control the secretion of diffusible factors that, in turn, induce cardiogenesis in mesoderm. At a molecular level, the diffusible factors controlled by these pathways are distinct. Aim 1 is to define novel diffusible molecules that are regulated by Dkk1 and directly induced cardiogenesis in mesoderm. These include both the molecules that are the induced by suppression the canonicalWnt/beta- catenin pathway by the carboxyl terminal cysteine-rich domain of Dkk1, as well as molecules regulated by a novel activity that we localized to the amino-terminal cysteine-rich domain. Aim 2 will evaluate molecules downstream of Nodal. Aim 3 is to characterize the synergy between the pathways involved in cardiogenesis to define the signals that govern progression to later stages of differentiation and tissue organization, including specification of myocyte contractility and heart tube morphogenesis. The signals that control embryonic cardiogenesis have been demonstrated to function in stem cell cardiogenesis; thus, elucidation of signaling molecules that act directly on competent mesoderm should enable regenerative technologies.